Glycobiology Advance Access originally published online on March 29, 2006
Glycobiology 2006 16(7):641-650; doi:10.1093/glycob/cwj103
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Endogenously produced ganglioside GM3 endows etoposide and doxorubicin resistance by up-regulating Bcl-2 expression in 3LL Lewis lung carcinoma cells
3 Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 21- Nishi 11, Kita-ku, Sapporo 001-0021, Japan; 4 Core Research for Evaluational Science and Technology Program (CREST), Japan Science and Technology Corporation (JST), Graduate School of Pharmaceutical Sciences, Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University, Kita 21-Nishi 11, Kita-ku, Sapporo 001-0021, Japan; 5 Department of Chemistry, Dong-Eui University, (San24, Gaya-dong) 995, Eomgwangno, Busan 614-714, South Korea; and 6 Pharmacodynamics, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan
1 To whom correspondence should be addressed; e-mail: jin{at}tohoku-pharm.ac.jp
2 Present address: Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan
Received on January 19, 2006; revised on March 7, 2006; accepted on March 17, 2006
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionMaterials and MethodsConflict of Interest StatementAcknowledgmentsReferences |
|---|
The ganglioside patterns have been shown to dramatically change during cell proliferation and differentiation and in certain cell-cycle phases, brain development, and cancer malignancy. To investigate the significance of the ganglioside GM3 in cancer malignancy, we established GM3-reconstituted cells by transfecting the cDNA of GM3 synthase into a GM3-deficient subclone of the 3LL Lewis lung carcinoma cell line (Uemura, S. (2003) Glycobiology, 13, 207–216). The GM3-reconstituted cells were resistant to apoptosis induced by etoposide and doxorubicin. There were no changes in the expression levels of topoisomerase II
or P-glycoprotein, or in the uptake of doxorubicin between the GM3-reconstituted cells and the mock-transfected cells. To understand the mechanism of the etoposide-resistant phenotype acquired in the GM3-reconstituted cells, we investigated their apoptotic signaling. Although no difference was observed in the phosphorylation of p53 at serine-15-residue site by etoposide between the GM3-reconstituted cells and mock-transfected cells, the activation of both caspase-3 and caspase-9 was specifically inhibited in the former. We found that the anti-apoptotic protein B-cell leukemia/lymphoma 2 (Bcl-2) was increased in the GM3-reconstituted cells. Moreover, wild-type 3LL Lewis lung carcinoma cells, which have an abundance of GM3, exhibited no DNA fragmentation following etoposide treatment and expressed higher levels of the Bcl-2 protein compared with the J5 subclone. Thus, these results support the conclusion that endogenously produced GM3 is involved in malignant phenotypes, including anticancer drug resistance through up-regulating the Bcl-2 protein in this lung cancer cell line. Key words: anticancer drugs / apoptosis / Bcl-2 / ganglioside GM3 / lung cancer
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of Interest StatementAcknowledgmentsReferences |
|---|
The expression of gangliosides and alterations in their composition have been observed during cell proliferation and differentiation and in certain cell-cycle phases, brain development, and cancer malignancy. Aberrant glycosylation is recognized as a common feature of cancer cells. It is thought to be a result of initial oncogenic transformation and a key event in the induction of invasion and metastasis. For these reasons, the relationship between cancer malignancy and the expression levels of gangliosides has been researched for many years (Hakomori, 2002
). The ganglioside GD3, for instance, has been demonstrated to promote tumor cell motility and growth, possibly through angiogenesis and vascular endothelial growth factor (VEGF) production (Zeng et al., 2000). Changes in ganglioside composition in colon cancer cells, caused by the enhanced expression of the ganglioside-specific sialidase (Neu3), were found to lead to an accumulation of lactosylceramide and to endow apoptosis resistance, mainly through increased B-cell leukemia/lymphoma 2 (Bcl-2) levels and decreased caspase expression (Kakugawa et al., 2002). Additionally, the ganglioside GM3, a major ganglioside in many mammalian cells, is abundantly expressed in highly metastatic cell lines of B16 melanoma (Yogeeswaran et al., 1978; Nozue et al., 1988). There have been other reports that suggest the involvement of gangliosides in apoptosis. GD3 was found to be actively synthesized and transiently accumulated in the early stages of apoptosis and to relocate to the mitochondrial membranes, where it causes the opening of the permeability transition pore complex (PTPC) and the release of apoptogenic factors (De Maria et al., 1997). In another report, GM3 overexpression in murine bladder cancer cells induced apoptosis and reduced their malignant potential, normally characterized by cell proliferation, motility, and invasion, as well as xenograft tumor growth (Watanabe et al., 2002). The results of some of these studies, however, are conflicting, which may be caused by the variety of cell lines and methods used. Therefore, significance of the biological functions of gangliosides in malignancy is still controversial.
There have been some studies on gangliosides and drug resistance. Doxorubicin-resistant small-cell lung cancer (SCLC) cell line SBC-3/ADM100 expressed higher level of GM3, and cisplatin-resistant SCLC cell line SBC-3/CDDP exhibited greater increase of GM3 compared with the parent cell line, suggesting that the alteration of ganglioside composition may be involved in the acquisition of drug resistance (Kiura et al., 1998).
Membrane microdomains are organized by glycosphingolipids together with cholesterol and sphingomyelin, where they modulate signal transduction through their effect on protein kinase activity. Some studies have demonstrated that several signal transducer molecules (e.g., c-Src, Ras, Rho, and focal adhesion kinase [FAK]) are enriched in the microdomains and can be co-immunoprecipitated with GM3, indicating that they are closely associated with the ganglioside (Iwabuchi, Handa, et al., 1998; Iwabuchi, Yamamura, et al., 1998).
The GM3 synthase (SAT-I) cDNA was identified in 1998 (Ishii et al., 1998), making the regulation of SAT-I cDNA expression possible. In a previous report, we established GM3-reconstituted cells by transfecting the SAT-I cDNA into J5 cells, a GM3-deficient clone of the murine 3LL Lewis lung carcinoma cell line, to study the biological function of the ganglioside (Uemura et al., 2003). Compared with the controls, GM3-reconstituted cells exhibit a greater ability to form colonies in soft agar and become resistant to apoptosis when serum is depleted, suggesting that endogenously produced GM3 might regulate tumor-progression abilities. In this study, we have examined the relationship between GM3 and anticancer drug resistance using these GM3-reconstituted lung cancer cells.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of Interest StatementAcknowledgmentsReferences |
|---|
J5/SAT-I cells showed resistance to anticancer drug etoposide
In our previous study, we established GM3-reconstituted cells (J5/SAT-I cells) by transfecting the SAT-I cDNA into J5 cells, a GM3-deficient clone of the murine 3LL Lewis lung carcinoma cell line. Compared with the controls, J5/SAT-I cells exhibit a greater ability to form colonies in soft agar and become resistant to apoptosis when serum is depleted, suggesting that endogenously produced GM3 might regulate tumor-progression abilities. To understand the relationship between GM3 and anticancer drug resistance, we examined sensitivity to anticancer drugs and apoptosis in these GM3-reconstituted lung cancer cells.
The viability of GM3-reconstituted cells (J5/SAT-I cells) was significantly higher than that of negative vector-transfected cells (mock cells), following exposure to increasing concentrations of the topoisomerase II inhibitors etoposide and doxorubicin for 24 h (Figure 1A and B). The IC50 values of etoposide in J5/SAT-I cells and mock cells were 114.4 3.104 µM and 3.350 0.091 µM, respectively, and the resistance index was 34.1-fold (Table I). Though the resistance index of doxorubicin was 3.80-fold, J5/SAT-I cells exhibited greater resistance to doxorubicin in the growth-inhibition curves (Table I, Figure 1B).
|
, mock; 
, J5/SAT-I. (C) DNA fragmentation. Cells were exposed to etoposide at increasing concentrations for 24 h, and the cellular DNA was extracted and resolved by 1.5% agarose gel electrophoresis. Lane M shows a 100 bp DNA marker. (D) Analysis of annexin V and propidium iodide staining by flow cytometry. After exposure to 10 µM etoposide for 24 h, cells were collected and washed, then stained by annexin V–FITC and propidium iodide, and analyzed by flow cytometry. The horizontal and vertical axes indicate annexin V and propidium iodide staining, respectively.
|
To reveal the mechanism of etoposide resistance in J5/SAT-I cells, we analyzed cell death in the J5/SAT-I and mock cells by two independent methods, examining the DNA degradation for the "laddering" pattern typical of apoptosis (Figure 1C) and annexin V staining, which detects the translocated phosphatidylserine in the outer membrane that indicates an early stage of apoptosis (Figure 1D). In the DNA fragmentation analysis, etoposide treatment (5–100 µM) induced apoptosis only in the mock cells, correlating with drug concentration (Figure 1C). DNA fragmentation was not detected in J5/SAT-I cells, though the cells were treated with 100 µM etoposide.
As shown in Figure 1D, etoposide-induced cell death was also assessed by annexin V and propidium iodide staining as analyzed by flow cytometry. Cells were treated with 10 µM etoposide for 24 h and then detected. Our results indicate that annexin V-positive and propidium iodide-negative cells, which indicate an early stage of apoptosis, were fewer in J5/SAT-I cells (0.90%) compared with mock cells (17.80%) when treated with etoposide. From Figure 1C and D, it has been suggested that J5/SAT-I cells showed more resistance to apoptosis compared with mock cells.
No differences were observed in P-glycoprotein expression and intracellular doxorubicin concentrations between J5/SAT-I and mock cells
P-glycoprotein-modulated drug efflux is the most widely characterized drug-resistance mechanism. To investigate changes in drug uptake and efflux in J5/SAT-I cells, we determined the expression of P-glycoprotein and measured the concentration of intracellular doxorubicin (Figure 2). First, we determined the expression level of P-glycoprotein by western blotting (Figure 2A). It has been previously reported that human colon cancer cell line and its doxorubicin-resistant sub-line, SW620 Ad300, showed different expression levels of P-glycoprotein (Lai et al., 1991). As the latter expressed high level of P-glycoprotein, we chose these two cell lines for negative and positive control, respectively. As shown in Figure 2A, only SW620 Ad300 cell line showed P-glycoprotein expression. No expression of P-glycoprotein was detected in both the J5/SAT-I and the mock cells as well as in SW620 cell line. Second, we measured intracellular doxorubicin concentrations in these four cell lines to analyze the differences in drug uptake (Figure 2B). Cells were treated with 0, 0.25, 0.50, and 1 µM doxorubicin for 24 h, and then intracellular doxorubicin concentrations were determined. Although SW620 Ad300 cells had a significantly lower accumulation of doxorubicin compared with the parent SW620 cells, there was no difference in the intracellular accumulation of this drug between J5/SAT-I and mock cells (Figure 2B), suggesting no alteration in drug uptake and efflux in J5/SAT-I cells. Therefore, acquisition of drug resistance might occur intracellularly.
|
The death receptor-mediated apoptosis was slightly delayed in J5/SAT-I cells
Activation of apoptosis signaling following treatment with cytotoxic drugs, including anticancer drugs, has been shown to lead to activation of the mitochondrial (intrinsic) pathway of apoptosis. On the other hand, signaling through the death receptor (extrinsic) pathways contributes to the sensitivity of cells, such as TNF-, toward cytotoxic treatments. These pathways converge, finally, at the level of the caspases, the effector molecules in most forms of cell death. Caspases have been implicated in apoptosis induced by various kinds of anticancer drugs; hence, we examined their involvement in etoposide-induced apoptosis in mock cells, using the caspase inhibitor Z-aspartyl-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB). Pretreatment with Z-Asp-CH2-DCB inhibited etoposide-induced DNA fragmentation in mock cells, confirming that the apoptosis was caspase-dependent (data not shown).
Next, to analyze which apoptosis-signaling pathway is inhibited in J5/SAT-I cells, we investigated apoptosis induced by TNF-, using DNA fragmentation analysis (Figure 3A). Although the mock cells treated with TNF- showed more DNA ladder formation than J5/SAT-I cells, TNF- induced apoptosis in both cells, even in the presence or absence of the protein-synthesis inhibitor cycloheximide (CHX). The presence of CHX enhanced the sensitivity of TNF- in both cell lines. As shown in Figure 3B, TNF- induced DNA fragmentation from the sixth hour in mock cells, whereas from the ninth hour in J5/SAT-I cells. Although a 3-h delay was observed in J5/SAT-I cells, TNF- induced DNA fragmentation in both cell lines. Pretreatment of the caspase inhibitor Z-Asp-CH2-DCB inhibited TNF--induced DNA fragmentation in both cells (Figure 3A), showing that the apoptosis was caspase-dependent. Therefore, we examined the death receptor signaling after treatment of TNF- by measuring activation of caspase-8 and caspase-3 (Figure 3C and D). After treatment of TNF- in the presence of CHX for 6 h, caspase-8 activity of mock and J5/SAT-I cells was 359 and 173%, respectively (Figure 3C). Caspase-8 activity was highly increased (2.08-fold) in mock cells compared with J5/SAT-I cells. We also confirmed that these activities were inhibited by the presence of caspase-8 inhibitor IETD-FMK (data not shown). Although cleaved caspase-3 was detected from the sixth hour in both cell lines, J5/SAT-I cells showed lower level of cleaved caspase-3 compared with mock cells, and these results were consistent with the fact that DNA fragmentation was strongly observed from the sixth hour in mock cells, whereas J5/SAT-I cells showed it from the ninth hour (Figure 3B–D). These results indicated that J5/SAT-I cells showed a slight resistance to receptor-induced apoptosis; however, the apoptosis resistance in J5/SAT-I cells was mainly through the mitochondrial pathway.
|
The activation of caspase-3 and caspase-9 by etoposide treatment was inhibited in J5/SAT-I cells
From Figure 3, it is suggested that resistance to apoptosis in J5/SAT-I cells is mainly through the mitochondrial pathway. To further examine this possibility and to determine the altered step in the apoptosis pathway in J5/SAT-I cells, we investigated the activation of caspase-9 and caspase-3, which is related to the mitochondrial pathway, and the phosphorylation of p53. Many groups have shown that phosphorylation of p53 at serine-15 residue (p53-Ser15) occurs after DNA damage, and the phosphorylation of this site is involved in the activation of p53 (Shieh et al., 1997; Prives and Hall, 1999). Following treatment with etoposide, J5/SAT-I and mock cells exhibited phosphorylation of p53-Ser15, as early as 1 h (Figure 4). However, although the active forms of caspase-9 and caspase-3 were detected in mock cells after 12 h, no activation was detected in J5/SAT-I cells. There were no differences, though, in the protein levels of the inactive forms of caspase-9, caspase-3 (full length of caspase-9 and caspase-3), and p53 between the two cell lines (data not shown). These data suggest that the apoptosis-signaling pathway might be inhibited at the upstream of the activation of caspase-9, which lies farthest upstream, in J5/SAT-I cells, though DNA damage was caused by etoposide in both cell lines.
|
Anti-apoptotic protein Bcl-2 expression was up-regulated in J5/SAT-I clones
To examine which pathway might be altered at the upstream of caspase-9, we next investigated the expression of major Bcl-2 family proteins, which are known to regulate apoptosis through the mitochondrial pathway. The protein levels of the pro-apoptotic factors—Bax, Bak, Bcl-XS, and Bad—and the anti-apoptotic factor—Bcl-XL—were similar in J5/SAT-I and mock cells (Figure 5A). However, the protein level of the anti-apoptotic protein Bcl-2 was increased in two J5/SAT-I clones (Figure 5A, upper panel). -Tubulin was determined for internal control. To assess whether the up-regulation of the Bcl-2 protein was due to an increase in mRNA expression, we performed a quantitative real-time PCR analysis. There was no difference between the mock and J5/SAT-I cells in the expression of Bcl-2 mRNA (Figure 5B). Thus, these results suggest that Bcl-2 protein was up-regulated by post-translational modification in J5/SAT-I cells.
|
No differences were detected in the expression levels of topoisomerase II, an etoposide target molecule
Etoposide resistance has been linked to quantitative and qualitative alterations of topoisomerase II proteins in some cases (Robert and Larsen, 1998). To test the mechanism of etoposide resistance in GM3-reconstituted cells, topoisomerase II protein levels were measured by western blotting (Figure 6). No difference in the protein levels of topoisomerase II was observed between J5/SAT-I and mock cells.
|
A slight increase of ceramide content was observed in etoposide-treated J5/SAT-I cells
There have been many studies that indicate ceramide is a mediator of apoptosis, and it has been reported that serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis (Hannun, 1996; Perry et al., 2000). Therefore, we measured the ceramide levels in J5/SAT-I cells treated with etoposide. Etoposide treatment elevated ceramide levels in J5/SAT-I cells at 24 h, yet there was no change in the mock cells (Figure 7). From these results, we concluded that a decrease in ceramide levels might not contribute to apoptosis-resistant phenotype in J5/SAT-I cells.
|
Wild-type (parent) 3LL Lewis lung carcinoma cells showed resistance to etoposide with high levels of GM3 and Bcl-2 expression
To assess the generality, we examined whether the phenomena, in which GM3-reconstituted J5/SAT-I cells showed resistance to etoposide and expressed higher levels of Bcl-2 protein, could also be observed in other GM3-rich cell lines. DNA fragmentation analysis was assessed on wild-type (parent) 3LL Lewis lung carcinoma cells containing high levels of GM3 and the J5 clone, which lacks GM3 (Inokuchi et al., 1993; Figure 8A). Although etoposide induced DNA fragmentation in J5 clone, the parent 3LL cells showed resistance to etoposide-induced apoptosis (Figure 8A). Moreover, the Bcl-2 protein levels were measured in these two cell lines by western blotting, and it was found that the parent 3LL cells also displayed high levels of the Bcl-2 protein compared with the J5 clone (Figure 8B). These results demonstrated a correlation between GM3 expression and anticancer drug resistance in the 3LL Lewis lung carcinoma cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of Interest StatementAcknowledgmentsReferences |
|---|
In our previous study, we established GM3-reconstituted cells (J5/SAT-I) by transfecting the SAT-I gene into J5 cells, a GM3-deficient clone of the murine 3LL Lewis lung carcinoma cell line (Uemura et al., 2003
). J5/SAT-I cells acquired greater ability to form colonies in soft agar than mock cells, indicating that endogenous GM3 is essential for maintaining fundamental properties of malignant cells. In this study, we examined the relationship between GM3 expression and anticancer drug resistance in J5/SAT-I cells. J5/SAT-I cells showed resistance to etoposide-induced apoptosis assessed by DNA fragmentation analysis and flow cytometry. No alteration in doxorubicin uptake and efflux was observed in J5/SAT-I cells. On the other hand the activation of caspase-9 and caspase-3 after etoposide treatment was inhibited in J5/SAT-I cells. Therefore, the acquisition of anticancer drug resistance by transfecting the SAT-I cDNA into murine lung cancer cells is mainly due to enhanced expression of anti-apoptotic protein Bcl-2. It is well known that cancer cells have varying responses to anticancer drugs. Elucidation of cellular-resistance mechanisms holds the promise of better treatment strategies for cancer patients. Our results indicate that J5/SAT-I cells exhibited resistance to doxorubicin and especially greater resistance to etoposide, whose common mechanism is the inhibition of topoisomerase II. We endeavored to reveal the mechanisms involved in this distinct drug resistance.
Drug-resistance mechanisms involve a variety of factors—the actions of the multi-drug efflux protein such as P-glycoprotein, the quantitative and qualitative alteration of target proteins, the alteration of drug metabolism, the activation of the ability to repair DNA double-strand breaks, aberrance of apoptosis-signaling pathways, and so on (Kruh, 2003). We found no differences in the expression of P-glycoprotein or in the uptake of doxorubicin (Figure 2). These data imply that the acquisition of selective drug resistance against etoposide and doxorubicin in J5/SAT-I cells was not due to a multi-drug efflux mechanism but rather appeared to involve intracellular factors. As shown in Figures 1 and 3, apoptosis-signaling pathway was mainly inhibited in mitochondrial pathway in J5/SAT-I cells. The early stage of the cell-stress response, indicated by p53 phosphorylation, did occur in both mock and J5/SAT-I cells (Figure 4). However, the activation of both caspase-9 and caspase-3 was selectively attenuated in the J5/SAT-I cells, indicating that the apoptosis signaling could be inhibited upstream of caspase-9.
The p53 tumor-suppressor protein is stabilized and activated by a variety of cellular stresses including DNA damage, which in turn leads to growth arrest and/or apoptosis (Fridman and Lowe, 2003). Phosphorylation of p53-Ser15 is known to occur after DNA damage, leading to stabilization and activation of the protein (Shieh et al., 1997; Prives and Hall, 1999). Reportedly, in mouse embryo fibroblasts, a serine to alanine mutation in the p53-Ser18 (corresponding to human p53-Ser15) attenuated the apoptotic response, indicating that phosphorylation of this site is a contributing factor for apoptosis (Woo et al., 2002). Our results indicate that there was no difference in the serine phosphorylation of p53 following etoposide treatment, implying that the two cell lines have similar DNA damage after uptake of etoposide (Figure 4).
In light of the inhibition of the caspase-9 activation pathway in GM3-reconstituted cells, we investigated the expression of major Bcl-2 family proteins known to regulate this pathway. We found that the expression of the Bcl-2 protein in steady-state cells was increased in the GM3-reconstituted cells (Figure 5A). Bcl-2 and its anti-apoptotic homologues guard mitochondrial membrane integrity until neutralized by a Bcl-2 homology (BH3) only protein. Bax and Bak then form homo-oligomers within the mitochondrial membrane, resulting in the release of cytochrome c, which activates Apaf-1, allowing it to bind to and activate caspase-9. It has been suggested that Bcl-2 is a well-known inhibitor of apoptosis by association with pro-apoptotic proteins Bax and Bak, and studies with gene-targeted cells indicated that the presence of Bax or Bak is required for many forms of apoptosis, and each type of cell needs at least one of the anti-apoptotic Bcl-2 family members to survive (Cory and Adams, 2002; Yang and Yu, 2003). Bax protein levels were not up-regulated after p53 was phosphorylated in etoposide-treated cells (data not shown). Protein levels of Bcl-2 were increased, whereas those of Bax and Bak were not changed in J5/SAT-I cells, suggesting that the ratio of Bcl-2 to Bax/Bak was significantly increased; thus, consequent imbalance between Bcl-2 and Bax/Bak can inhibit apoptosis in J5/SAT-I cells (Figure 5A).
There are some reports of the relationship between gangliosides and Bcl-2. It has been reported that transfection of the sialidase (Neu3) cDNA into human colon cancer cells inhibited apoptosis and was accompanied by increased Bcl-2 expression (Kakugawa et al., 2002). Ganglioside GD3 can induce apoptosis through mitochondrial pathway (De Maria et al., 1997), and the forced expression of Bcl-2 significantly prevents GD3-induced apoptosis (Rippo et al., 2000; Simon et al., 2002). In addition, recent studies showed that GD3 synthase has an apoptotic effect on vascular endothelial ECV304 cells through down-regulation of Bcl-2 expression via dephosphorylation of AKT and CREB (Ha et al., 2004). In our studies, the anti-apoptotic Bcl-2 protein levels were increased in J5/SAT-I cells, indicating that Bcl-2 is up-regulated by post-translational modification in J5/SAT-I cells. Bcl-2 family members are controlled through transcription, phosphorylation, proteolytic cleavage, and ubiquitination and proteasome degradation systems (Yang and Yu, 2003). It will be necessary to investigate whether the up-regulation of Bcl-2 is caused by elevated translation from mRNA or increased protein stability, including its ubiquitination and any proteasome involvement.
Etoposide and doxorubicin are DNA topoisomerase inhibitors and form a DNA–topoisomerase–drug complex known as the cleavable complex (Larsen and Skladanowski, 1998). Recent reports identified the histone H1.2 as a cytochrome c-releasing factor that plays an important role in transmitting apoptotic signals from the nucleus to the mitochondria, following DNA double-strand breaks (Konishi et al., 2003). There are some reports suggesting that the levels of protein involved in double-strand break repair, namely DNA-dependent protein kinase (DNA-PKcs) and RAD51, are up-regulated in the etoposide-resistant human small-cell lung cancer cell line CPH 54B, which does, in fact, exhibit a high repair rate of double-strand breaks (Hansen, Lundin, Helleday, et al., 2003; Hansen, Lundin, Spang-Thomsen, et al., 2003). Our result indicates that there was no difference in topoisomerase II protein level between mock and J5/SAT-I cells (Figure 6); however, it will be important to investigate the DNA repair mechanism, target protein alteration, and metabolism of anticancer drugs to categorize the spectrum of resistance in J5/SAT-I cells and, perhaps, to find a clue to reveal the mechanism of drug resistance.
It is increasingly apparent that sphingolipids, and in particular ceramide, are important mediators in regulating the stress response (Hannun, 1996). One report indicated that serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis (Perry et al., 2000). In our study, etoposide treatment induced a slight elevation of ceramide levels in J5/SAT-I cells compared with the mock cells (Figure 7). This result indicated that ceramide-mediated apoptosis is not a major mechanism involved in drug resistance in J5/SAT-I cells.
To verify the generality of the phenomena, in which GM3-reconstituted J5/SAT-I cells showed resistance to etoposide and expressed higher levels of Bcl-2 protein, we examined the etoposide-induced DNA fragmentation and the Bcl-2 protein level in the steady state in the wild-type (parent) 3LL Lewis lung carcinoma cells containing GM3-rich and GM3-deficient cell J5 clone (Figure 8). The 3LL cells showed resistance to etoposide-induced apoptosis and expressed higher levels of the Bcl-2 protein compared with the J5 clone. These results imply the fact that the GM3-expression level is important in sensitivity to etoposide in some cases of lung carcinoma cells.
Taken together, our results demonstrate that endogenously produced GM3 could up-regulate Bcl-2 protein, suggesting a novel drug-resistance mechanism by GM3 in these lung carcinoma cells. One of the conceivable reasons of drug-resistance acquisition in J5/SAT-I cells is that certain proteins, which interact with GM3, have changed its function caused by the overexpression of GM3. The other possibility is that the balance of sphingolipids might be important to maintain homeostasis; thus, change in sphingolipid composition might induce drug resistance in our J5/SAT-I cells. Various cell lines have different compositions of gangliosides; thus, change of ganglioside patterns by transfecting the same gene may cause numerous patterns of gangliosides, and it might depend on cell type. Moreover, change of ganglioside contents can cause change of pares of signaling molecules which interact with GM3, and it might be cell-type dependent. These possibilities might be the reason of the fact that results from some reports focused on GM3 function are not always coincident. In this viewpoint, factors that regulate Bcl-2 expression level might change by transfecting SAT-I cDNA in the case of J5/SAT-I cells. We are in the process of examining the subcellular localization of GM3 and its target molecules that are related to the acquisition of etoposide and doxorubicin drug resistance. One approach will be a detailed analysis of signal transduction from GM3-enriched membrane microdomains. Finally, it will be necessary to investigate post-translational modification of Bcl-2 and other apoptosis-relating proteins in J5/SAT-I cells to elucidate the molecular mechanism of anti-apoptotic effect of GM3. We are currently ensuring whether human lung cancers show correlation between GM3 expression levels and anticancer drug resistance to apply these findings to therapeutical use such as diagnosis in the future.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of Interest StatementAcknowledgmentsReferences |
|---|
Materials
Etoposide, doxorubicin hydrochloride, CHX (all from Sigma-Aldrich, St. Louis, MO), recombinant human TNF-
(Techne Corporation, Minneapolis, MN), and Z-Asp-CH2-DCB (Bachem, Heidelberg, Germany) were used for apoptosis assays.
Cell cultures
The J5 subclone of the murine 3LL Lewis lung carcinoma cell line has been described previously (Inokuchi et al., 1993; Uemura et al., 2003). Cells were maintained in RPMI 1640 medium (Sigma) containing 10% (v/v) fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) 100 units/mL penicillin, and 100 µg/mL streptomycin (Sigma). Cells were cultured in a humidified 5% CO2 atmosphere and passaged every 3 days. SAT-I-transfected cells (J5/SAT-I cells) and vector-transfected cells (mock cells) were cultured in the above medium containing 300 µg/mL zeocin (Invitrogen, Carlsbad, CA). The human colon carcinoma SW620 cell line and its doxorubicin-resistant derivative SW620 Ad 300 were generous gifts of Dr. Kamo (Lai et al., 1991; Komatsubara et al., 1999). These cell lines were maintained in RPMI 1640 medium as described above. SW620 Ad 300 cells were cultured in medium supplemented with 300 ng/mL doxorubicin.
Cytotoxicity assays
Drug cytotoxicity was determined by using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Briefly, the day before treatment, cells in RPMI 1640 containing 10% FBS and 300 µg/mL zeocin at 1 x 105 cells/mL were plated in 96-well microtiter plates at 100 µL/well. After the cells had been exposed to various concentrations of etoposide and doxorubicin or control buffer for 24 h, 10 µL of WST-8 reagent was added to each well, and the cells were then incubated for 2 h at 37°C. The absorbance at 450 nm of the formazan generated in the wells was measured with a dual-wavelength flying spot scanner (CS9300-PC, Shimazu, Kyoto, Japan).
DNA fragmentation assay
DNA fragmentation assay was performed by using the method previously reported by Ito and others with some modification (Ito et al., 1999). Briefly, cells were treated with each drug for 24 or 48 h and then collected. Cells were washed with phosphate-buffered saline (PBS) and lysed in buffer (10 mM Tris–HCl [pH 7.4], 10 mM ethylenediaminetetraacetic acid [EDTA], and 0.5% Triton X-100) for 10 min at 4°C and centrifuged at 20,000x g for 15 min. The supernatant was incubated with 200 µg/mL RNase A for 1 h at 37°C, followed by treatment with 200 µg/mL proteinase K for 30 min at 50°C. The DNA was extracted with equal volumes of phenol, phenol/chloroform (1:1, v/v), and chloroform and then precipitated with a 0.2 volume of 5 M NaCl and a 1.2 volume of 2-propanol. The DNA was suspended in 10 mM Tris–HCl (pH 7.4) and 1 mM EDTA and its concentration determined by the absorbance at 260 nm. Each 20 µg sample of DNA was separated by electrophoresis on a 1.5% agarose gel in 40 mM Tris–HCl (pH 8.5) and 2 mM EDTA. The gel was then stained with 0.5 µg/mL ethidium bromide for 15 min, and the fragmented DNA was visualized under UV light and photographed.
Cell death detection by flow cytometry
In this assay, we used MEBCYTO-Apoptosis Kit (Medical & Biological Laboratories, Nagoya, Japan) and followed the manufacturer’s instructions. Briefly, cells were cultured in 6-well culture dish and treated with 10 µM etoposide for 24 h. After apoptosis was induced, cells were collected and washed once with PBS, resuspended in binding buffer, and then stained with annexin V–FITC and propidium iodide at room temperature for 15 min in the dark. The intensity of cell fluorescence was determined by a FACScan cytometer (Becton Dickinson, La Jolla, CA).
Caspase-8 activity assay
Caspase-8 activity was measured by using APOCYTO Caspase-8 Colorimetric Assay Kit (Medical & Biological Laboratories). Briefly, cells were treated with 10 ng/mL TNF- in the presence of 1 mg/mL CHX for 6 h. After treatment of TNF-, cells were collected and counted, then centrifuged at 400x g for 10 min, resuspended in cell lysis buffer, and incubated on ice for 10 min. After centrifugation at 10,000x g for 5 min, supernatants were transferred to new microcentrifuge tubes, and total protein concentration of the cell extracts was measured by using BCA Protein Assay kit (Pierce Chemical Company, Rockford, IL). Reaction buffer with DTT mix and caspase-8 substrate were added to each sample and then incubated at 37°C for 4 h. Finally, samples were measured at 405 nm in a spectrophotometer.
Immunoblotting
Cells were lysed in lysis buffer (50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 2 mM NaF, 1 mM EDTA, 1 mM ethylene glycol bis(2-aminoethyl ether)-tetraacetic acid, 1% Triton, 1 mM phenylmethylsulfonyl fluoride, 75 U/mL aprotinin, 10 µg/mL leupeptin, 10 µg/mL pepstatin, and 1 mM sodium orthovanadate) for 10 min at 4°C. Protein concentrations were determined with a BCA Protein Assay kit (Pierce Chemical Company, Rockford, IL). Equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). Membranes were blocked for 1 h with blocking buffer (Tris-buffered saline containing 0.05% Tween-20 and 5% skimmed milk), then incubated for 1 h with a primary antibody diluted in the same solution. Anti-Bcl-XS/L, anti-Bax, anti-Bak, anti-topoisomerase II (all from Santa Cruz Biotechnology, Santa Cruz, CA), anti-caspase-3, anti-caspase-9, anti-phospho-p53 (Ser15) (all from Cell Signaling Technology, Beverly, MA), anti-Bcl-2 (Oncogene Research Products, Cambridge, MA), anti-P-glycoprotein (Dako Corporation, Carpinteria, CA), and anti--tubulin (Sigma) were used as primary antibodies. After three washes, the blot was incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Horseradish peroxidase-conjugated anti-mouse, anti-rabbit, and anti-goat IgG F(ab')2 fragments (all from Amersham Biosciences, Buckinghamshire, UK) were used as secondary antibodies. Labeling was detected using ECLTM Reagents, ECL Plus Western blotting detection reagent (both from Amersham Biosciences), and Lumi-LightPLUS Western blotting substrate (Roche Diagnostics, Mannheim, Germany).
Measurement of intracellular doxorubicin concentrations
For determination of the intracellular doxorubicin concentrations, the method previously reported by Itoh and others was used with some modification (Itoh et al., 2003). Briefly, cells were treated with 0, 0.25, 0.50, and 1 µM doxorubicin for 24 h and then collected. For determination of the intracellular doxorubicin concentrations, aliquots of cell suspensions were cooled on ice and then centrifuged at 150x g for 3 min. The cells were washed and resuspended in ice-cold PBS, then mixed for 30 s with five volumes of chloroform/methanol (4 : 1, v/v) and centrifuged at 1200x g for 15 min. The concentration of doxorubicin in the organic phase was determined with a Shimazu RF-1500 spectro-fluorophotometer (Shimazu, Kyoto, Japan; excitation, 470 nm; emission, 585 nm).
Real-time quantitative PCR
Total RNA was extracted from cultured cells using the TRIZOL reagent (Invitrogen) following the instructions of the manufacturer. cDNA was prepared from 2.5 µg total RNA using first-strand cDNA synthesis kit for reverse transcription–polymerase chain reaction (RT–PCR) (AMV) (Roche Diagnostics, Penzberg, Germany) according to the manufacturer’s protocol. Real-time PCR was performed using a standard TaqMan PCR kit protocol on an Applied Biosystems 7500 Real Time PCR System (Applied Biosystems, Foster City, CA). TaqMan universal PCR master mix, primers, and probes of rodent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as well as mouse Bcl-2 were purchased from Applied Biosystems. The 50 µL PCR mixtures include 5 µL RT product, 2x TaqMan Universal PCR Master Mix, 0.25 µM TaqMan probe, 0.9 µM forward primer, and 0.9 µM reverse primer. The reactions were incubated in a 96-well plate at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, and at 60°C for 1 min. All reactions were run in triplicate.
DGK assay
Ceramide levels in total lipid extracts of GM3-reconstituted cells were measured using a modified version of the diacylglycerol kinase (DGK) assay of Preiss and others (Preiss et al., 1986). Briefly, cells (1 x 107) were treated with etoposide for the indicated times and then collected, washed twice with PBS. The total lipids were extracted from the cells with chloroform/methanol/water (4:4:0.3 and 2:4:0.3), successively, 1.94 ml of 10 mg/ml DOPG (L--dioleoyl-phosphatidylglycerol) was dried up and resuspended in 1 ml of 7.5% ß-OG (Octyl ß-D-glucopyranoside) in water to prepare ß-OG/DOPG mixed micelles. Total lipids were resuspended in 20 µL of ß-OG/DOPG mixed micelles by sonication and vortexing vigorously. 20 µL micellar lipids was added to a reaction mixture containing 0.2 µL DTT (1 M), 2 µL E. coli DGK (7.1 U/mL), and 1 µL [
-32P] ATP (10 mCi/mL in tricine buffer, pH 6.7) in 50 µL reaction buffer (100 mM imidazole [pH 6.6], 100 mM LiCl, 25 mM MgCl2, and 2 mM EGTA [pH 6.6]), and 17.8 µL dilution buffer (100 mM imidazole [pH 6.6] with 1 mM diethylenetriaminepentaacetic acid). After incubating for 30 min at 37°C, lipids were extracted with 0.6 mL chloroform/methanol (1 : 1, v/v). After vortexing the mixture, 265 µL of 1 M KCl was added, and the phases were separated by centrifugation. An aliquot of the organic phase was dried, resuspended with 20 µL chloroform, and spotted onto a high-performance thin layer chromatography (HPTLC) plate (Merck, Darmstadt, Germany). Lipids were separated in chloroform/acetone/methanol/acetic acid/water (10 : 4 : 3 : 2 : 1). Radioactive bands were visualized with an imaging analyzer (BAS-2000; Fuji Film, Kanagawa, Japan), and 32P-ceramide-1-phosphate was quantitated.
|
Conflict of Interest Statement |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of Interest StatementAcknowledgmentsReferences |
|---|
None declared.
|
Acknowledgments |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of Interest StatementAcknowledgmentsReferences |
|---|
We express our deep appreciation to Dr. Uehara for providing anti-Bcl-XS/L antibody and valuable comments. This study was supported in part by grants-in-aid 15390019 (to J.I.) and 15040202 (Scientific Research on Priority Area to J.I.) from the Ministry of Education, Culture, Sports, Science and Technology. This work was also supported by the Mizutani Foundation for Glycoscience (to J.I.).
|
Abbreviations |
|---|
Bcl-2, B-cell leukemia/lymphoma 2; ß-OG, Octyl ß-d-glucopyranoside; CHX, cycloheximide; DGK, diacylglycerol kinase; DOPG, l-
-dioleoyl-phosphatidylglycerol; EDTA, ethylenediaminetetraacetic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p53-Ser15, p53 at serine-15 residue; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SAT-I, GM3 synthase; SCLC, small-cell lung cancer; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TNF-, tumor necrosis factor-alpha; Z-Asp-CH2-DCB, Z-aspartyl-2,6-dichlorobenzoyloxymethylketone |
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of Interest StatementAcknowledgmentsReferences |
|---|
Cory, S. and Adams, J.M. (2002) The Bcl2 family: regulators of the cellular life-or-death switch. Nat. Rev. Cancer, 2, 647–656.[CrossRef][Web of Science][Medline]
De Maria, R., Lenti, L., Malisan, F., d’Agostino, F., Tomassini, B., Zeuner, A., Rippo, M.R., and Testi, R. (1997) Requirement for GD3 ganglioside in CD95- and ceramide-induced apoptosis. Science, 277, 1652–1655.
Fridman, J.S. and Lowe, S.W. (2003) Control of apoptosis by p53. Oncogene, 22, 9030–9040.[CrossRef][Web of Science][Medline]
Ha, K.T., Lee, Y.C., and Kim, C.H. (2004) Overexpression of GD3 synthase induces apoptosis of vascular endothelial ECV304 cells through downregulation of Bcl-2. FEBS Lett., 568, 183–187.[Medline]
Hakomori, S. (2002) Glycosylation defining cancer malignancy: new wine in an old bottle. Proc. Natl. Acad. Sci. U.S.A., 99, 10231–10233.
Hannun, Y.A. (1996) Functions of ceramide in coordinating cellular responses to stress. Science, 274, 1855–1859.
Hansen, L.T., Lundin, C., Helleday, T., Poulsen, H.S., Sorensen, C.S., Petersen, L.N., and Spang-Thomsen, M. (2003) DNA repair rate and etoposide (VP16) resistance of tumor cell subpopulations derived from a single human small cell lung cancer. Lung Cancer, 40, 157–164.[CrossRef][Web of Science][Medline]
Hansen, L.T., Lundin, C., Spang-Thomsen, M., Petersen, L.N., and Helleday, T. (2003) The role of RAD51 in etoposide (VP16) resistance in small cell lung cancer. Int. J. Cancer, 105, 472–479.[CrossRef][Web of Science][Medline]
Inokuchi, J., Jimbo, M., Kumamoto, Y., Shimeno, H., and Nagamatsu, A. (1993) Expression of ganglioside GM3 and H-2 antigens in clones with different metastatic and growth potentials isolated from Lewis lung carcinoma (3LL) cell line. Clin. Exp. Metastasis, 11, 27–36.[CrossRef][Web of Science][Medline]
Ishii, A., Ohta, M., Watanabe, Y., Matsuda, K., Ishiyama, K., Sakoe, K., Nakamura, M., Inokuchi, J., Sanai, Y., and Saito, M. (1998) Expression cloning and functional characterization of human cDNA for ganglioside GM3 synthase. J. Biol. Chem., 273, 31652–31655.
Ito, A., Uehara, T., Tokumitsu, A., Okuma, Y., and Nomura, Y. (1999) Possible involvement of cytochrome c release and sequential activation of caspases in ceramide-induced apoptosis in SK-N-MC cells. Biochim. Biophys. Acta, 1452, 263–274.[Medline]
Itoh, M., Kitano, T., Watanabe, M., Kondo, T., Yabu, T., Taguchi, Y., Iwai, K., Tashima, M., Uchiyama, T., and Okazaki, T. (2003) Possible role of ceramide as an indicator of chemoresistance: decrease of the ceramide content via activation of glucosylceramide synthase and sphingomyelin synthase in chemoresistant leukemia. Clin. Cancer Res., 9, 415–423.
Iwabuchi, K., Handa, K., and Hakomori, S. (1998) Separation of "glycosphingolipid signaling domain" from caveolin-containing membrane fraction in mouse melanoma B16 cells and its role in cell adhesion coupled with signaling. J. Biol. Chem., 273, 33766–33773.
Iwabuchi, K., Yamamura, S., Prinetti, A., Handa, K., and Hakomori, S. (1998) GM3-enriched microdomain involved in cell adhesion and signal transduction through carbohydrate-carbohydrate interaction in mouse melanoma B16 cells. J. Biol. Chem., 273, 9130–9138.
Kakugawa, Y., Wada, T., Yamaguchi, K., Yamanami, H., Ouchi, K., Sato, I., and Miyagi, T. (2002) Up-regulation of plasma membrane-associated ganglioside sialidase (Neu3) in human colon cancer and its involvement in apoptosis suppression. Proc. Natl. Acad. Sci. U.S.A., 99, 10718–10723.
Kiura, K., Watarai, S., Ueoka, H., Tabata, M., Gemba, K., Aoe, K., Yamane, H., Yasuda, T., and Harada, M. (1998) An alteration of ganglioside composition in cisplatin-resistant lung cancer cell line. Anticancer Res., 18, 2957–2960.[Web of Science][Medline]
Komatsubara, M., Nagata, T., Miyauchi, S., and Kamo, N. (1999) Cimetidine enhances the antiproliferative effect of 5-fluorouracil on colon carcinoma SW620. Anticancer Res., 19, 1153–1157.[Web of Science][Medline]
Konishi, A., Shimizu, S., Hirota, J., Takao, T., Fan, Y., Matsuoka, Y., Zhang, L., Yoneda, Y., Fujii, Y., Skoultchi, A.I., and Tsujimoto, Y. (2003) Involvement of histone H1.2 in apoptosis induced by DNA double-strand breaks. Cell, 114, 673–688.[CrossRef][Web of Science][Medline]
Kruh, G.D. (2003) Introduction to resistance to anticancer agents. Oncogene, 22, 7262–7264.[CrossRef][Web of Science][Medline]
Lai, G.M., Chen, Y.N., Mickley, L.A., Fojo, A.T., and Bates, S.E. (1991) P-glycoprotein expression and schedule dependence of adriamycin cytotoxicity in human colon carcinoma cell lines. Int. J. Cancer, 49, 696–703.[Web of Science][Medline]
Larsen, A.K. and Skladanowski, A. (1998) Cellular resistance to topoisomerase-targeted drugs: from drug uptake to cell death. Biochim. Biophys. Acta, 1400, 257–274.[Medline]
Nozue, M., Sakiyama, H., Tsuchiya, K., Hirabayashi, Y., and Taniguchi, M. (1988) Melanoma antigen expression and metastatic ability of mutant B16 melanoma clones. Int. J. Cancer, 42, 734–738.[Web of Science][Medline]
Perry, D.K., Carton, J., Shah, A.K., Meredith, F., Uhlinger, D.J., and Hannun, Y.A. (2000) Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis. J. Biol. Chem., 275, 9078–9084.
Preiss, J., Loomis, C.R., Bishop, W.R., Stein, R., Niedel, J.E., and Bell, R.M. (1986) Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells. J. Biol. Chem., 261, 8597–8600.
Prives, C. and Hall, P.A. (1999) The p53 pathway. J. Pathol., 187, 112–126.[CrossRef][Web of Science][Medline]
Rippo, M.R., Malisan, F., Ravagnan, L., Tomassini, B., Condo, I., Costantini, P., Susin, S.A., Rufini, A., Todaro, M., Kroemer, G., and Testi, R. (2000) GD3 ganglioside directly targets mitochondria in a bcl-2-controlled fashion. FASEB J., 14, 2047–2054.
Robert, J. and Larsen, A.K. (1998) Drug resistance to topoisomerase II inhibitors. Biochimie, 80, 247–254.[Medline]
Shieh, S.Y., Ikeda, M., Taya, Y., and Prives, C. (1997) DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell, 91, 325–334.[CrossRef][Web of Science][Medline]
Simon, B.M., Malisan, F., Testi, R., Nicotera, P., and Leist, M. (2002) Disialoganglioside GD3 is released by microglia and induces oligodendrocyte apoptosis. Cell Death Differ., 9, 758–767.[CrossRef][Web of Science][Medline]
Uemura, S., Kabayama, K., Noguchi, M., Igarashi, Y., and Inokuchi, J. (2003) Sialylation and sulfation of lactosylceramide distinctly regulate anchorage-independent growth, apoptosis, and gene expression in 3LL Lewis lung carcinoma cells. Glycobiology, 13, 207–216.
Watanabe, R., Ohyama, C., Aoki, H., Takahashi, T., Satoh, M., Saito, S., Hoshi, S., Ishii, A., Saito, M., and Arai, Y. (2002) Ganglioside GM3 overexpression induces apoptosis and reduces malignant potential in murine bladder cancer. Cancer Res., 62, 3850–3854.
Woo, R.A., Jack, M.T., Xu, Y., Burma, S., Chen, D.J., and Lee, P.W. (2002) DNA damage-induced apoptosis requires the DNA-dependent protein kinase, and is mediated by the latent population of p53. EMBO J., 21, 3000–3008.[CrossRef][Web of Science][Medline]
Yang, Y. and Yu, X. (2003) Regulation of apoptosis: the ubiquitous way. FASEB J., 17, 790–799.
Yogeeswaran, G., Stein, B.S., and Sebastian, H. (1978) Altered cell surface organization of gangliosides and sialylglycoproteins of mouse metastatic melanoma variant lines selected in vivo for enhanced lung implantation. Cancer Res., 38, 1336–1344.
Zeng, G., Gao, L., Birkle, S., and Yu, R.K. (2000) Suppression of ganglioside GD3 expression in a rat F-11 tumor cell line reduces tumor growth, angiogenesis, and vascular endothelial growth factor production. Cancer Res., 60, 6670–6676.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Prinetti, M. Aureli, G. Illuzzi, S. Prioni, V. Nocco, F. Scandroglio, N. Gagliano, G. Tredici, V. Rodriguez-Menendez, V. Chigorno, et al. GM3 synthase overexpression results in reduced cell motility and in caveolin-1 upregulation in human ovarian carcinoma cells Glycobiology, January 1, 2010; 20(1): 62 - 77. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Roque-Navarro, K. Chakrabandhu, J. de Leon, S. Rodriguez, C. Toledo, A. Carr, C. M. de Acosta, A.-O. Hueber, and R. Perez Anti-ganglioside antibody-induced tumor cell death by loss of membrane integrity Mol. Cancer Ther., July 1, 2008; 7(7): 2033 - 2041. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||